The objectives of this proposal are two-fold: 1) to provide the chlamydial research community with a genetic tool kit (based upon a mobile, targetable group II intron [TargeTron, Sigma]) that will allow us to tackle questions not before feasible, and 2) to use these tools to test the essentiality of the polymorphic outer membrane proteins (Pmps), a collection of phase variable chlamydial proteins linked to virulence. To address our first goal, we will expand the utility of the TargeTron system by enabling its use in different Chlamydia species and C. trachomatis serovars through the development of non-ampicillin based selection and screening markers. In addition, we will modify this platform for use in gene editing and random mutagenesis approaches. In Aim 2, we seek to determine the essentiality of C. trachomatis pmps using the optimal intron-platform developed in Aim 1 to target each pmp for insertional inactivation. These autotransporter proteins are conserved throughout Chlamydia spp. and have been associated with virulence in humans. Elucidating which Pmps (C. trachomatis possesses nine pmps) are required for growth will allow future research efforts to focus on those Pmps critical for pathogen survival. Collectively, our studies will provide a powerful set of molecular tools to help advance research aimed at vaccine development and treatment strategies, while also filling a knowledge gap regarding the importance of Pmp function to chlamydial pathogenesis.